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Gonadotropin-Releasing Hormone Neurons Express Estrogen Receptor-ß

Laboratory of Endocrine Neurobiology (E.H., I.K., Z.L.), Institute of Experimental Medicine, Hungarian Academy of Sciences; Department of Forensic Medicine (N.S., E.K.), Semmelweis University; and Department of Neuroscience (Z.L.), Faculty of Information Technology, Pázmány Péter Catholic University, Budapest, 1083 Hungary; and Departments of Epidemiology and Preventive Medicine and Anatomy and Neurobiology (I.M.), University of Maryland, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Erik Hrabovszky, M.D., Ph.D., Department of Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, 1083 Hungary. E-mail: hrabovszky{at}koki.hu.

	Abstract

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Context: Recent identification of the second estrogen receptor (ER) isoform (ER-ß) within GnRH neurons of the rodent brain has generated much enthusiasm in the field of neuroendocrine research by questioning the dogma that GnRH cells do not directly sense changes in circulating estrogens.

Objective: To address the issue of whether GnRH neurons of the human hypothalamus also contain ER-ß, we have performed dual-label immunocytochemical studies.

Design: Tissue sections were prepared from autopsy samples of male human individuals (n = 8; age < 50 yr), with sudden causes of death. Technical efforts were made to minimize postmortem interval (<24 h), optimize tissue fixation (use of a mixture of 2% paraformaldehyde and 4% acrolein for four tissue samples), and sensitize the immunocytochemical detection of ER-ß (application of silver-intensified nickel-diaminobenzidine chromogen).

Main Outcome Measure: Distribution and percent ratio of GnRH neurons that also contained ER-ß immunoreactivity were analyzed under the light microscope.

Results: With acrolein in tissue fixative, nuclear ER-ß immunoreactivity was observed in 10.8–28.0% of GnRH neurons of the four different individuals. ER-ß-containing GnRH neurons were widely distributed in the hypothalamus, without showing a noticeable preference in regional location.

Conclusions: The demonstration of ER-ß and the previous lack of detection of ER- in human GnRH cells indicate that estrogens may exert direct actions upon GnRH neurons exclusively through ER-ß. In the light of differing ligand-binding characteristics of ER-ß from those of ER-, this discovery offers a potential new approach to influence estrogen feedback to GnRH neurons through ER-ß-selective receptor ligands.

	Introduction

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THE GnRH (ALSO CALLED LHRH) neurosecretory system represents the final common hypothalamic pathway in the neuroendocrine control of reproduction. Changing levels of the ovarian sex steroid hormone 17ß-estradiol (E2) tightly regulate the activity of the neuroendocrine reproductive axis through feedback actions to GnRH cells (for review, see Ref. 1). In 1983, Shivers et al. (2) reported that GnRH neurons of the rat do not accumulate tritiated E2 in vivo. This surprising observation and the coherent results of subsequent immunocytochemical studies that failed to show any estrogen receptors (ERs) in GnRH neurons (3, 4) suggested that estrogen signaling is communicated to the GnRH neuronal system by estrogen-sensitive interneurons and/or glial cells. Shortly after the discovery of a second ER isoform (ER-ß), this view was challenged by reports of ER- immunoreactivity (5), ER- mRNA (6), ER-ß mRNA (6, 7), and [¹²⁵I]estrogen-binding sites (7) in rodent GnRH neurons. In 2000, our group published the results of dual-label in situ hybridization experiments that showed absence of ER- and presence of ER-ß mRNA hybridization signal in GnRH neurons of rats (7). This report was followed by the withdrawal (8) of previous ER- mRNA detection in mouse GnRH neurons (6). The opinion that rodent GnRH neurons only contain the ER-ß isoform started to form. In 2001, two groups detected nuclear ER-ß immunoreactivity in 63–73% of GnRH neurons in the rat (9, 10). These immunocytochemical data, together with the previous observation that GnRH neurons of the rat are capable of accumulating a ¹²⁵I-labeled estrogen compound in vivo (7), strengthened the concept that GnRH neurons synthesize functional ER-ß.

The recent demonstration of immunoreactive ER-ß in a large subset of ovine GnRH neurons (11) that lack ER- (12) indicates that the selective presence of ER-ß in GnRH cells is not restricted to rodent species. The issue of whether or not GnRH neurons of the human also contain ER-ß remained unresolved. Therefore, in the present immunocytochemical study, we addressed the putative presence of ER-ß in GnRH neurons of the human hypothalamus.

	Materials and Methods

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Tissue samples

Human hypothalamic samples from eight male individuals with postmortem intervals between 12–14 h were obtained at autopsy from the Forensic Medicine Department of Semmelweis University (Budapest, Hungary) using protocols reviewed and approved by the Regional Committee of Science and Research Ethics (TUKEB 49/1999). Selection criteria included sudden causes of death, lack of history of neurological and endocrinological disorders, postmortem intervals between 12–24 h and age less than 50 yr. Hypothalamic blocks were dissected out according to guidelines of the optic chiasm (rostrally), mammillary bodies (caudally), the anterior commissure (dorsally), and 2 cm lateral from the midsagittal plane (laterally). The tissue blocks were initially rinsed for 5 min in several changes of 4% paraformaldehyde solution prepared with 0.1 M PBS (pH 7.4) and then transferred for 2 d into a freshly prepared mixture of 4% acrolein and 2% paraformaldehyde (4 C).

Section preparation

Tissue blocks were infiltrated with 30% sucrose for 5 d and cut in half in the midsagittal plane to reduce section size. The hemi-hypothalami were aligned in a freezing mold, surrounded with Jung tissue freezing medium (Leica Microsystems, Nussloch Gmbh, Germany; diluted 1:1 with 0.9% sodium chloride solution), frozen on powdered dry ice, and sectioned serially at 30 µm with a Leica CM 3050 S cryostat (Leica) according to the plane of the lamina terminalis. The sections were stored in antifreeze solution (30% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) at –20 C until used.

Dual-label immunocytochemical studies

Every 20th section from each block was processed for dual-label immunocytochemistry. Sections were pretreated with 1% sodium borohydride (30 min), a mixture of 0.5% H₂O₂ and Triton X-100 (30 min), 3.7% paraformaldehyde in 30% ethanol (30 min), 8% thioglycolic acid (30 min), and 2% normal horse serum (30 min). The sections were first incubated in a 1:20,000 dilution (in 2% normal horse serum) of primary ER-ß antibodies (P3; gift from Dr. P. T. Saunders, Edinburgh, UK) raised in sheep and targeting the A/B region of human ER-ß (13), followed by biotinylated secondary antibodies (Jackson ImmunoResearch Europe Ltd., Soham, Cambridgeshire, UK; 1:1,000) and ABC Elite reagent (60 min each). The signal was visualized with nickel-intensified diaminobenzidine and then post-intensified with silver-gold (9, 10). Subsequently, GnRH immunoreactivity was detected with rabbit primary antibodies (LR-1, 1:20,000; gift from Dr. R. A. Benoit, Montreal, Canada) using the biotinylated secondary antibody-ABC technique and nonintensified diaminobenzidine as chromogen. The dual-labeled sections were mounted on microscope slides and coverslipped with DPX mounting medium (Fluka Chemie, Buchs, Switzerland).

	Results

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Distribution of ER-ß immunoreactivity in the human hypothalamus

The immunocytochemical detection of ER-ß caused nuclear immunolabeling in hypothalamic (Fig. 1A) neurons. The silver-gold intensification step enhanced the staining of labeled nuclei and rendered the nickel-diaminobenzidine chromogen dark and granular (Figs. 1A and 2, A–G). No immunoreactivity was observed if the primary or secondary antibodies were omitted or when the ER-ß antibody had been immunoneutralized with 5 µg/ml of the immunization antigen (Fig. 1B). Increasing dilutions of the primary antibody led to a commensurate attenuation of the immunoreactive signal.

View larger version (49K): [in this window] [in a new window]   FIG. 1. Detection of ER-ß-immunoreactive neurons in the human hypothalamus. The ER-ß antigen was visualized with the P3 antibodies using the silver-gold intensification of the nickel-diaminobenzidine peroxidase reaction product (black). A, Immunolabeling was localized to the cell nuclei, was abundant in the dorsolateral (SODL) and ventromedial subdivisions (SOVM) of the supraoptic nucleus, but also occurred in high numbers in other hypothalamic regions, including the lateral hypothalamic area (LHA). Note that after the immunolocalization of ER-ß, this section also went through the immunocytochemical detection of GnRH using diaminobenzidine chromogen. The two GnRH neurons in boxed regions exhibited ER-ß labeling in their cell nucleus, as shown at high magnification in Fig. 2, A and B. B, Section showing the lack of any ER-ß signal went through the immunocytochemical detection of ER-ß using a primary antiserum preabsorbed with 5 µg/ml of the immunization antigen. Scale bar, 100 µm.

View larger version (103K): [in this window] [in a new window]   FIG. 2. Demonstration of ER-ß immunoreactivity in GnRH neurons of the human hypothalamus. The ER-ß antigen was detected using the silver-gold intensification of nickel-diaminobenzidine peroxidase reaction product (black), whereas GnRH neurons were stained with the nonintensified diaminobenzidine chromogen (brown). Cell nuclei immunoreactive for ER-ß occurred in high numbers in the lateral hypothalamic area (LHA) (A and B), ventromedial nucleus (VMH) (C and D), medial basal hypothalamus (MBH) (C and E), infundibular nucleus (INF) (F), and lateral tuberal nucleus (LTU) (F and G). The GnRH neurons were scattered within the same regions and often contained ER-ß-immunoreactive nucleus. Note two dual-labeled GnRH neurons in the LHA (A and B; also shown in boxed regions of Fig. 1A) that are bipolar in shape. Inset in B demonstrates the characteristic nuclear labeling for ER-ß. Single-labeled (E) and double-labeled (D and G) GnRH neurons are both found in the MBH (D) and the LTU (G), marked with boxes in low-power images C and F. Some GnRH cells exhibit a multipolar morphology (E and G). Scale bars, 100 µm (C and F), 25 µm (A, B, D, E, and G), and 5 µm (high-power inset in B).

Identification of ER-ß-immunoreactive GnRH neurons

GnRH neurons occurred in very low numbers within individual hypothalamic sections (Figs. 1A and 2C). Bipolar (Fig. 2B) and multipolar (Fig. 2G) neuronal shapes were distinguishable. High-power microscopic analysis of acrolein/paraformaldehyde-fixed sections revealed ER-ß in a subset of GnRH neurons (Fig. 2, B, D, and G), whereas other GnRH cells contained no signal for ER-ß (Fig. 2E). Dual-labeled neurons occurred in highest numbers in the lateral and dorsal hypothalamic areas, in the infundibular, lateral tuberal, ventromedial, periventricular, supraoptic, and paraventricular nuclei, without showing a preferential location in any particular hypothalamic region (Fig 2). Their percent ratio in the four individuals was 10.8, 11.1, 21.7, and 28.0%, respectively. Paraformaldehyde fixation, in itself, was not sufficient to visualize any ER-ß immunoreactivity in GnRH neurons.

	Discussion

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Results of this dual-label immunocytochemical study provide the first evidence for ER-ß in human GnRH neurons. This observation, together with the previously reported lack of ER- in these cells (15) indicate that E2 may directly regulate human GnRH neurons via the ER-ß receptor isoform.

The use of postmortem tissue samples in this study raises the question as to what extent suboptimal tissue processing compromised the sensitivity of ER-ß detection. Paraformaldehyde fixation, in itself, was clearly incompatible with the visualization of any ER-ß immunoreactivity in the cell nuclei of GnRH cells. Similarly to this observation on human samples, the inclusion of acrolein in the fixative was critically important to maximize the number of ER-ß-immunoreactive GnRH neurons in the rat (9, 10). On the other hand, paraformaldehyde-based fixation of human hypothalamic tissue samples clearly ensured sufficient antigen preservation to visualize ER-ß immunoreactivity in magnocellular neurosecretory neurons (14). The uncontrolled tissue damage before tissue fixation could interfere further with the visualization of ER-ß in GnRH cells of postmortem human tissues. This could account for the variations in the intensity of ER-ß staining and in the percentage (10.8–28.0%) of the ER-ß-immunoreactive GnRH neurons across the four acrolein/paraformaldehyde-fixed samples. These technical considerations suggest that the ratio of ER-ß-expressing GnRH neurons could be significantly underestimated in this study.

A physiological correlate of our present studies is the use of male hypothalami. This has eliminated differences in the ER-ß staining of GnRH neurons as an effect of variations in circulating estrogen levels. It is worth noting that our previous immunocytochemical study on rats, indeed, revealed a negative impact of E2 on the percent ratio of ER-ß-immunoreactive GnRH neurons (10). Given that only male individuals were included in this study, the potential sexual dimorphism of the colocalization phenomenon will also require future clarification with the parallel use of similarly processed tissue samples from men and women. In this context, it is worth mentioning that previous in situ hybridization (9) and immunocytochemical (9) studies failed to detect any significant gender difference in the percent ratio of ER-ß-synthesizing GnRH neurons in rats. Although results of preliminary studies also indicate the presence of ER-ß in GnRH neurons of the human female, the low sample number has not allowed yet a systematic assessment of potential sex differences (Hrabovszky, E., unpublished observations).

The results of the present immunocytochemical experiments provide strong evidence for the presence of ER-ß in human GnRH neurons. In the light of previous studies that showed the lack of ER- mRNA in human GnRH neurons (15) and the accumulating evidence that only the ER-ß isoform occurs in GnRH neurons of rodents (7, 8) and ewes (11), it is reasonable to speculate that direct estrogen actions upon human GnRH neurons may be exerted exclusively via ER-ß. With this information, the growing number of ER-ß-selective ligands may offer a unique tool to modulate GnRH cell functions and thereby the human reproductive axis. This long-term perspective necessitates a solid knowledge about the direct actions of ER-ß on GnRH cell functions. Several studies on rodent GnRH neurons have already identified such ER-ß-mediated direct estrogen actions. The fast, nongenomic actions of ligand-bound ER-ß include rapid phosphorylation of cAMP-response element-binding protein (16) and stimulation of intracellular calcium oscillations (17) in mouse GnRH neurons. Estrogen also increases excitability of mouse GnRH neurons, partly via mechanisms that include phosphorylation of potassium channels (18). In vitro transfection experiments have also established the role of ER-ß in a ligand-independent transcriptional activation and a ligand-dependent transcriptional repression of the mouse GnRH promoter (19). Furthermore, in vivo studies with newly available ER-ß-selective agonist ligands provided evidence for the ER-ß-mediated induction of galanin mRNA expression in GnRH neurons of rats (20). Although the precise role of ER-ß in fertility of laboratory animals and humans remains to be determined, one has to remain aware of well-established indirect estrogen actions that involve interneurons expressing the ER- isoform.

In summary, we have identified ER-ß immunoreactivity in GnRH neurons of the human hypothalamus. Because these cells lacked ER- in previous studies, the new findings indicate that estrogens may exert direct actions upon GnRH cells selectively through the ER-ß isoform. The differing ligand-binding characteristics of ER-ß from those of the classical ER- offers a potential new strategy to influence estrogen feedback mechanisms to human GnRH neurons via recently available ER-ß-selective ligands.

	Acknowledgments

 
We thank Drs. R. A. Benoit and P. T. Saunders for kindly providing the LR1 (GnRH) and P3 (ER-ß) antibodies, respectively, and E. Dobó for helpful technical advice.

	Footnotes

 
This work was supported by grants from the National Science Foundation of Hungary (OTKA T43407 and T46574), NKFP 1A/002/2004, and the European Union FP6 funding (LSHM-CT-2003-50304).

This publication reflects the authors’ views and not necessarily those of the European Union. The information in this document is provided as is, and no guarantee or warranty is given that the information is fit for any particular purpose. The user thereof uses the information at its sole risk and liability.

Disclosure: All of the authors have nothing to declare.

First Published Online April 24, 2007

¹ E.H. and I.K. have contributed equally to the studies presented in this manuscript.

Abbreviations: E2, 17ß-Estradiol; ER, estrogen receptor.

Received December 20, 2006.

Accepted April 16, 2007.

	References

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